PROJECT SUMMARY. Modern synthetic organic chemistry methodologies and internet `how-to' websites have likely made it impossible to prevent the presence of synthetic psychoactive agents in our society. Determining the mechanism of action (MOA) of central nervous system (CNS) drugs is a cumbersome task but is essential to better treat substance abuse and episodic toxicity. On a fundamental level, psychoactive drugs must bind to biological targets to elicit an effect. This work aims to develop a platform with the ultimate goal of studying ligand-receptor interactions in live animals to unravel the MOA of modern psychoactive agents. Photoaffinity labeling (PAL) has been used to successfully map ligand binding domains for several receptor and drug classes because PAL probes covalently attach to all binding partners upon transient radiation with UV light. However, the use of PAL has been restricted to cell culture or tissue homogenates because most animals have endogenous molecules capable of blocking penetration of UV light. Pigment free zebrafish, known as casper fish, have been developed which completely lack pigmentation and may therefore be useful for in vivo PAL studies. The creation of a PAL derivative of a psychoactive agents may allow us to identify binding partners at the same time that drug induced behavioral changes are observed via transient UV radiation and follow-up bioconjugation chemistry to determine protein modification. This small grant has two aims. We will synthesize a well-defined library of PAL probes to examine the structure activity relationship of methamphetamines and MDMA. Probes will utilize an arylazide photoreactive group and a terminal alkyne for conjugation with biochemical reporters- a fluorophore for visualization. Aim 2 will involve PAL method development. This will include defining behavioral dose-response in casper zebrafish (effective dose [ED50]). Once ED50's are defined, PAL will be performed as follows: incubate casper zebrafish with probe (at ED50) ? observe behavioral response ? irradiate with UV light ? dissect out and homogenize brain ? click to fluorophore ? SDS PAGE ? observe protein modification. Follow-up competition experiments and PAL background controls will validate findings. The experimental design above may allow us, for the first time, to observe direct binding partners responsible for behavioral changes associated with psychoactive agents. Upon establishing the feasibility of this approach we will seek to develop a larger probe library based on MDMA, methamphetamines, and novel synthetic cathinones (i.e. bath salts) in an attempt to correlate binding partners with toxicity, hallucinogenic effects, and psychostimulatory activity.